WldS requires Nmnat1 enzymatic activity and N16–VCP interactions to suppress Wallerian degeneration

Slow Wallerian degeneration (WldS) encodes a chimeric Ube4b/nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) fusion protein that potently suppresses Wallerian degeneration, but the mechanistic action of WldS remains controversial. In this study, we characterize WldS-mediated axon protection in vivo using Drosophila melanogaster. We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than WldS, and enzyme-dead versions of Nmnat1 and WldS exhibit severely reduced axon-protective function. Interestingly, a 16–amino acid N-terminal domain of WldS (termed N16) accounts for the differences in axon-sparing activity between WldS and Nmnat1, and N16-dependent enhancement of Nmnat1-protective activity in WldS requires the N16-binding protein valosin-containing protein (VCP)/TER94. Thus, WldS-mediated suppression of Wallerian degeneration results from VCP–N16 interactions and Nmnat1 activity converging in vivo. Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from WldS. Thus, nuclear Nmnat activity does not appear to be essential for WldS-like axon protection.

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WldS protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice

The Journal of Cell Biology ◽

10.1083/jcb.200807175 ◽

2009 ◽

Vol 184 (4) ◽

pp. 491-500 ◽

Cited By ~ 75

Author(s):

Laura Conforti ◽

Anna Wilbrey ◽

Giacomo Morreale ◽

Lucie Janeckova ◽

Bogdan Beirowski ◽

...

Keyword(s):

Nicotinamide Adenine Dinucleotide ◽

Wallerian Degeneration ◽

Adenosine Triphosphatase ◽

Chimeric Protein ◽

Biochemical Property ◽

Terminal Sequence ◽

Protective Function ◽

Nicotinamide Mononucleotide ◽

Binding Sequence

The slow Wallerian degeneration (WldS) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70–amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide–synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of WldS-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the WldS VCP-binding domain with an alternative ataxin-3–derived VCP-binding sequence restores its protective function. Enzyme-dead WldS is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. WldS requires both of its components to protect axons from degeneration.

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A novel polymorphism of human gene has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

Clinical Pharmacology & Therapeutics ◽

10.1016/j.clpt.2004.08.014 ◽

2004 ◽

Vol 76 (6) ◽

pp. 519-527 ◽

Cited By ~ 61

Author(s):

T FUKAMI ◽

M NAKAJIMA ◽

R YOSHIDA ◽

Y TSUCHIYA ◽

Y FUJIKI ◽

...

Keyword(s):

Amino Acid ◽

Enzymatic Activity ◽

Amino Acid Substitution ◽

Human Gene

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Differential requirements for FcγR engagement by protective antibodies against Ebola virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911842116 ◽

2019 ◽

Vol 116 (40) ◽

pp. 20054-20062 ◽

Cited By ~ 10

Author(s):

Stylianos Bournazos ◽

David J. DiLillo ◽

Arthur J. Goff ◽

Pamela J. Glass ◽

Jeffrey V. Ravetch

Keyword(s):

Ebola Virus ◽

Structural Constraints ◽

Global Public Health ◽

Protective Activity ◽

Protective Function ◽

Multiple Strategies ◽

Animal Disease Models ◽

Protective Antibodies ◽

Effector Activity

Ebola virus (EBOV) continues to pose significant threats to global public health, requiring ongoing development of multiple strategies for disease control. To date, numerous monoclonal antibodies (mAbs) that target the EBOV glycoprotein (GP) have demonstrated potent protective activity in animal disease models and are thus promising candidates for the control of EBOV. However, recent work in a variety of virus diseases has highlighted the importance of coupling Fab neutralization with Fc effector activity for effective antibody-mediated protection. To determine the contribution of Fc effector activity to the protective function of mAbs to EBOV GP, we selected anti-GP mAbs targeting representative, protective epitopes and characterized their Fc receptor (FcγR) dependence in vivo in FcγR humanized mouse challenge models of EBOV disease. In contrast to previous studies, we find that anti-GP mAbs exhibited differential requirements for FcγR engagement in mediating their protective activity independent of their distance from the viral membrane. Anti-GP mAbs targeting membrane proximal epitopes or the GP mucin domain do not rely on Fc–FcγR interactions to confer activity, whereas antibodies against the GP chalice bowl and the fusion loop require FcγR engagement for optimal in vivo antiviral activity. This complexity of antibody-mediated protection from EBOV disease highlights the structural constraints of FcγR binding for specific viral epitopes and has important implications for the development of mAb-based immunotherapeutics with optimal potency and efficacy.

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Structure and expression of the Drosophila ubiquitin-52-amino-acid fusion-protein gene

Biochemical Journal ◽

10.1042/bj2860281 ◽

1992 ◽

Vol 286 (1) ◽

pp. 281-288 ◽

Cited By ~ 10

Author(s):

H L Cabrera ◽

R Barrio ◽

C Arribas

Keyword(s):

Drosophila Melanogaster ◽

Amino Acid ◽

Fusion Protein ◽

Ribosomal Protein ◽

Protein Gene ◽

Genomic Library ◽

Regulatory Region ◽

Ribosomal Protein Genes ◽

Common Features ◽

Drosophila Cells

Ubiquitin belongs to a multigene family. In Drosophila two members of this family have been previously described. We report here the organization and expression of a third member, the DUb52 gene, isolated by screening a Drosophila melanogaster genomic library. This gene encodes an ubiquitin monomer fused to a 52-amino acid extension protein. There are no introns interrupting the coding sequence. Recently, it has been described that this extension encodes a ribosomal protein in Saccharomyces, Dictyostelium, and Arabidopsis. The present results show that the 5′ regulatory region of DUb52 shares common features with the ribosomal protein genes of Drosophila, Xenopus and mouse, including GC- and pyrimidine-rich regions. Moreover, sequences similar to the consensus Ribo-box in Neurospora crassa have been identified. Furthermore, a sequence has been found that is similar to the binding site for the TFIIIA distal element factor from Xenopus laevis. The DUb52 gene is transcribed to a 0.9 kb mRNA that is expressed constitutively throughout development and is particularly abundant in ovaries. In addition, the DUb52 gene has been found to be preferentially transcribed in exponentially growing Drosophila cells.

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Amino Acid Polymorphism and Rare Electrophoretic Variants of G6PD From Natural Populations of Drosophila melanogaster

Genetics ◽

10.1093/genetics/143.1.401 ◽

1996 ◽

Vol 143 (1) ◽

pp. 401-406

Author(s):

Walter F Eanes ◽

Michele Kirchner ◽

Daniel R Taub ◽

Jeanne Yoon ◽

Jiang-tian Chen

Keyword(s):

Drosophila Melanogaster ◽

Amino Acid ◽

Quaternary Structure ◽

Rare Variants ◽

Protein Domain ◽

Allozyme Polymorphism ◽

Electrophoretic Variants ◽

Function Classes ◽

Amino Acid Mutations

Abstract Identifing the amino acid changes responsible for electrophoretic variants is essential to understanding the significance of allozyme polymorphism in adaptation. The amino acid mutations responsible for the common G6PD allozyme polymorphisms in Drosophila melanogaster have been recently described. This study characterizes the amino acid changes associated with 11 rare electrophoretic G6PD variants. The 11 rare electrophoretic variants result from six independent amino acid mutations. The in vivo function of the rare variants was determined in an earlier study and most variants fell into one of two function classes. It is shown here that the function of the rare variants reflects the state of the Pro/Leu mutation responsible for the A / B allozyme polymorphism in each variant. Two mutations destabilize quaternary structure resulting in shifts from tetrameric to dimeric alleles, and one of these also results in a variant with in vivo function intermediate to A and B. That mutation is an aspartic-acid-to-asparagine change that is two residues away from the Pro/Leu polymorphism responsible for the A / B dimertetramer quaternary shift. Structure-function relationships based on studies of human G6PD deficency-associated mutations predict that these last two amino acid changes fall within the protein domain responsible for NADP-binding.

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Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching

10.1101/2020.01.20.913020 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tayah Hopes ◽

Michaela Agapiou ◽

Karl Norris ◽

Charley G.P. McCarthy ◽

Mary J O’Connell ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Amino Acid ◽

Ribosomal Protein ◽

Amino Acid Composition ◽

Acid Composition ◽

Mrna Translation

ABSTRACTRibosomes have long been thought of as homogeneous, macromolecular machines but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. Here, we have characterised ribosomal protein heterogeneity across 5 tissues of Drosophila melanogaster. We find that testes and ovaries contain the most heterogeneous ribosome populations, which occurs through paralog-switching. We have solved structures of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work allows us to propose that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation.

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Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury

Journal of Neuroinflammation ◽

10.1186/s12974-019-1679-x ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 4

Author(s):

Gerard Elberg ◽

Sigal Liraz-Zaltsman ◽

Fanny Reichert ◽

Takashi Matozaki ◽

Michael Tal ◽

...

Keyword(s):

Nerve Injury ◽

Wallerian Degeneration ◽

Axon Regeneration ◽

Recovery Of Function ◽

Regulatory Protein ◽

Wild Type ◽

Myelin Debris ◽

Dependent Inhibition ◽

Severed Axons

Abstract Background Recovery of function from traumatic nerve injury depends on the ability of severed axons to grow/regenerate back to their target tissues. This is achieved by successfully crossing the lesion site where physical impact severed axons, determined by the type of trauma, followed by successfully growing throughout the Wallerian degenerating nerve segment located distal to and beyond the lesion site, determined by the nature of Wallerian degeneration. The protracted removal of myelin debris in Wallerian degeneration, which leads residual myelin debris to slow down axon growth, impedes recovery of function. We focused in this study on mechanism(s) that delay the removal of myelin debris in Wallerian degeneration and so impede recovery. Previously, we showed that myelin debris inhibited its own phagocytosis in primary cultured macrophages and microglia as CD47 on myelin ligated SIRPα (signal regulatory protein-α) on phagocytes, and sequentially, SIRPα generated “don’t eat me” signaling. We also demonstrated that serum inhibited phagocytosis in a SIRPα-dependent manner. Herein, we aimed to determine whether SIRPα-dependent inhibition of phagocytosis in macrophages impedes the in vivo removal of myelin debris in Wallerian degeneration, further leading to impaired healing. Methods Using SIRPα null (SIRPα−/−) and littermate wild-type (SIRPα+/+) mice, we studied the recovery of sensory and motor functions from nerve injury and, further, axon regeneration, SIRPα expression, myelin debris removal, and the phagocytic capacity and presence of macrophages in Wallerian degeneration. Results Myelin debris removal, axon regeneration, and the recovery of functions were all faster in SIRPα−/− mice than in wild-type mice. Between the two cell types that mostly scavenge myelin debris, macrophages but not Schwann cells expressed SIRPα in wild-type mice, and furthermore, SIRPα−/− macrophages phagocytosed significantly more than wild-type macrophages. Conclusions Our findings suggest an intrinsic normally occurring SIRPα-dependent mechanism that impedes the in vivo removal of myelin debris in Wallerian degeneration by inhibiting the phagocytosis of myelin debris in macrophages, hence preventing fast growing axons from fully implementing their regenerative potential. Thus, accelerating the removal of myelin debris by eliminating SIRPα-dependent inhibition of phagocytosis will most likely advance recovery of functions from nerve injury.

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Regulation of a Drosophila melanogaster cGMP-specific phosphodiesterase by prenylation and interaction with a prenyl-binding protein

Biochemical Journal ◽

10.1042/bj20080560 ◽

2008 ◽

Vol 414 (3) ◽

pp. 363-374 ◽

Cited By ~ 6

Author(s):

Jonathan P. Day ◽

Vaughn Cleghon ◽

Miles D. Houslay ◽

Shireen-A. Davies

Keyword(s):

Drosophila Melanogaster ◽

Amino Acid ◽

Apical Membrane ◽

Binding Protein ◽

Membrane Targeting ◽

Type I ◽

S2 Cells ◽

Post Translational Modification ◽

Drosophila S2 Cells

Post-translational modification by isoprenylation is a pivotal process for the correct functioning of many signalling proteins. The Drosophila melanogaster cGMP-PDE (cGMP-specific phosphodiesterase) DmPDE5/6 possesses a CaaX-box prenylation signal motif, as do several novel cGMP-PDEs from insect and echinoid species (in CaaX, C is cysteine, a is an aliphatic amino acid and X is ‘any’ amino acid). DmPDE5/6 is prenylated in vivo at Cys1128 and is localized to the plasma membrane when expressed in Drosophila S2 cells. Site-directed mutagenesis of the prenylated cysteine residue (C1128S-DmPDE5/6), pharmacological inhibition of prenylation or co-expression of DmPrBP (Drosophila prenyl-binding protein)/δ each alters the subcellular localization of DmPDE5/6. Thus prenylation constitutes a critical post-translational modification of DmPDE5/6 for membrane targeting. Co-immunoprecipitation and subcellular-fractionation experiments have shown that DmPDE5/6 interacts with DmPrBP/δ in Drosophila S2 cells. Transgenic lines allow targeted expression of tagged prenylation-deficient C1128S-DmPDE5/6 in Type I (principal) cells in Drosophila Malpighian tubules, an in vivo model for DmPDE5/6 function. In contrast with wild-type DmPDE5/6, which was exclusively associated with the apical membrane, the C1128S-DmPDE5/6 mutant form was located primarily in the cytosol, although some residual association occurred at the apical membrane. Despite the profound change in intracellular localization of C1128S-DmPDE5/6, active transport of cGMP is affected in the same way as it is by DmPDE5/6. This suggests that, in addition to prenylation and interaction with DmPrBP/δ, further functional membrane-targeting signals exist within DmPDE5/6.

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Deletion of a Conserved Regulatory Element in the Drosophila Adh Gene Leads to Increased Alcohol Dehydrogenase Activity but Also Delays Development

Genetics ◽

10.1093/genetics/156.1.219 ◽

2000 ◽

Vol 156 (1) ◽

pp. 219-227 ◽

Cited By ~ 1

Author(s):

John Parsch ◽

Jacob A Russell ◽

Isabel Beerman ◽

Daniel L Hartl ◽

Wolfgang Stephan

Keyword(s):

Amino Acid ◽

Alcohol Dehydrogenase ◽

Enzymatic Activity ◽

Regulatory Element ◽

Catalytic Efficiency ◽

Purifying Selection ◽

Amino Acid Replacement ◽

Detectable Effect ◽

Twofold Increase

Abstract In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3′ UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3′ UTR sequence. Using transgenic experiments we show that deletion of the conserved 3′ UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3′ UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity.

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The influence of aromatic amino acids on the chemical composition of mint plants genus (Mentha L.)

Žemės ūkio mokslai ◽

10.6001/zemesukiomokslai.v28i1.4503 ◽

2021 ◽

Vol 28 (1) ◽

Author(s):

Aloyzas Velička ◽

Živilė Tarasevičienė ◽

Honorata Danilčenko

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Chemical Composition ◽

Aromatic Amino Acids ◽

Acid Solutions ◽

Protective Function ◽

Amino Acid Solutions ◽

Abiotic And Biotic Factors

The aromatic amino acids, tryptophan, phenylalanine and tyrosine, are not only components of proteins, but also precursors of many compounds in plants that have a significant impact on their growth, development, reproduction and protective function against various abiotic and biotic factors. With the growing demand for plant-derived chemical compounds, much in vitro and in vivo research is being conducted to intensify the synthesis of these compounds or to change their qualitative composition in plants. The aim of the research was to evaluate the influence of aromatic amino acids on the chemical composition of different varieties of Mentha L. plants. The field experiment was conducted at Aleksandras Stulginskis University (ASU) in 2017–2019, since 2019 at the Vytautas Magnus University Agricultural Academy Experimental Station, which is located in Ringaudai Eldership, Kaunas District (coordinates 54 ° 53′ 08.9″ N, 23° 50′ 08.02″ E). The effect of different concentrations of amino acid solutions on the chemical composition of mints depended on the mint variety. Spraying with all amino acids solutions significantly increased the dry matter, crude fiber and crude ash content in M. spicata ‘Crispa’ mints while a positive effect of amino acids solutions on the protein content was found only in M. piperita ‘Granada’ mints sprayed only with 200 mg l–1 tyrosine solution, where the amount of protein increased significantly by 1.41-fold compared to that of non-sprayed plants. The intensity of the synthesis of photosynthetic pigments depended on the amino acid solutions used and the variety of mint. There were both inhibitory and stimulatory effects.

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